A second member, erbB-2, in the erbB proto-oncogene family, had been identified based on gene amplification in a mammary carcinoma and close structural homology to the epidermal growth factor (EGF) receptor gene. Subsequently, overexpression in the presence or absence of gene amplification has been determined as the mechanism activating erbB-2 in human mammary tumor cell lines. These observations were paralleled by the finding that in vitro overexpression of the human erbB-2 coding sequence at protein levels similar to those observed in human mammary tumor cells was capable of conferring the transformed phenotype onto NIH/3T3 cells. Analysis of erbB-2 and EGF receptor protein levels in mammary tumor tissues revealed overexpression of erbB-2 in 45% (24/53) and EGF receptor of erbB-2 in 9% (4/47) of the patients analyzed. Gene amplification of erbB-2 and EGF receptor was associated with high protein expression levels in 19% and 4%, respectively, of the patients analyzed. Concordance of increased receptor gene expression in primary and metastatic lesions, combined with the observation that such alterations are detectable in mammary tumors as early as stages I and II, indicate that proto-oncogene activation resulting in the overexpression of growth factor receptor molecules can occur at a relatively early stage in the development of human mammary neoplasia. ErbB-3, a novel member of the erbB proto-oncogene family, was identified in normal genomic human DNA using v-erbB as a probe under reduced stringency hybridization conditions. Characterization of genomic exon sequences revealed structural homology to members of the tyrosine kinase family. The predicted amino acid sequence of these genomic exons indicated the closer homology of this region to the tyrosine kinase domains of the EGF receptor and erbB-2 protein than to any other known tyrosine kinase protein. Northern blot analysis using an exon-containing erbB-3 probe identified a 6.5kb gene-specific mRNA in normal and neoplastic cells of various tissue origin.